Allogeneic natural killer (NK) cells are gaining momentum as a cancer immunotherapy platform. Unlike autologous therapies, which require individualized manufacturing and long lead times, allogeneic NK products could offer “off-the-shelf” availability, reducing logistical and geographical barriers to treatment access.
A critical question for the field is identifying where NK cells deliver the greatest therapeutic benefit: can they serve as a primary modality for tumor elimination and sustained remission, or will they be a complementary treatment to other therapies? Emerging clinical data suggests that NK cells have the potential to achieve durable responses in certain hematological malignancies such as acute myeloid leukemia and acute lymphocytic leukemia (1,2).
Realizing this promise depends on overcoming manufacturing hurdles. Delivering consistent, highly functional NK cells at scale with compatible cryopreservation strategies will be essential to accelerate commercialization and accessibility.
In this article, we explore the current barriers to NK manufacturing, strategies to address them, and how innovative bioprocessing solutions are helping move the field closer to commercial readiness.
Key takeaways
- Allogeneic NK therapies offer off‑the‑shelf potential, helping overcome the logistical limitations of individualized autologous approaches with early clinical data shows promise for durable responses in hematologic cancers.
- Manufacturing consistency remains a critical barrier, with donor variability, feeder cell lines, and xeno‑derived components contributing to batch‑to‑batch variability that complicates optimization and regulatory compliance.
- High NK cell dose requirements intensify scale‑up challenges, putting pressure on manufacturers to adopt efficient expansion strategies capable of supporting allogeneic volumes.
- Automation, closed systems, and xeno‑free methods are emerging as key enablers of scalable, consistent NK manufacturing, making critical steps toward broad clinical accessibility.
How does NK cell therapy work?
Natural killer (NK) cells are a key part of the innate immune system, capable of rapidly identifying and destroying malignant cells without prior antigen sensitization. Their activity is regulated by a balance of activating and inhibitory receptors to detect stressed and abnormal cells while sparing healthy tissue.
Since NK cells don’t rely on antigen-specific receptors (unlike T cells), they are uniquely suited for allogeneic approaches. This factor reduces the risk of graft-versus-host disease (GvHD) while preserving graft-versus-tumor (GvT) activity—a critical advantage over autologous cell therapies. Early clinical trials have also indicated a therapeutically safer product with very low risk of complications such as cytokine release syndrome.
This favorable safety profile stems from NK cells innate biology and controlled cytokine secretion, making them an attractive option for patients who may not tolerate aggressive immunotherapies.
To harness this innate cytotoxicity for cancer treatment, NK cells must be expanded ex vivo to reach clinically effective doses and retain their functionality to the point of infusion into patients.
Why NK manufacturing is different
NK cell therapies face a unique challenge: very high cell doses per patient are needed due to their shorter persistence and limited in vivo proliferation. Obtaining sufficient numbers for clinical efficacy, especially when multiple infusions are needed, has become a major bottleneck. For example, doses for indications like multiple myeloma can reach up to 1.2 × 10⁷ NK cells per kilogram, yet NK cells represent only 5% to 20% of peripheral blood mononuclear cells (3).
Given these dosing requirements, efficient and scalable expansion strategies are essential. As an allogenic treatment, manufacturing processes must ultimately support large, consistent batches suitable for an off-the-shelf format that can treat multiple patients from a single production run.
Although an allogeneic therapy has yet to receive full FDA approval, several NK cell therapies are advancing through late-stage clinical trials. To realize off-the-shelf commercialization, the field must find strategies to consistently expand NK cells in large batches. Addressing this complexity is key to delivering on the promise of an accessible allogeneic immunotherapy.
Key challenges in NK cell therapy manufacturing
Managing variability
Batch-to-batch variability makes process optimization and standardization challenging, and the outcome of scale up difficult to predict. Without predictable process performance, progressing through clinical trials becomes nearly impossible. In NK cell manufacturing, much of this variability stems from the materials and reagents used during expansion.
Donor-derived starting material
Peripheral blood and umbilical cord blood are the most common sources for primary NK cells in clinical trials. While these materials are readily available and have demonstrated successful malignant cell targeting in preclinical and clinical studies, the inherent donor-to-donor variability can lead to inconsistent yields. Major clinical trials have already been negatively impacted by the heterogeneity of healthy patient donors (4).
Feeder cell lines and regulatory risk
Because NK cells make up only a small fraction of blood components, many clinical-stage NK cell therapies rely on feeder cell lines to stimulate the high level of expansion needed for therapeutic doses. The K562 cell line, derived from human leukemia cells, is the most well-characterized example, offering a difficult-to-match magnitude and speed of NK cell expansion (5).
These feeder cells must be irradiated post-expansion to prevent the introduction of dividing tumorigenic cells into a patient’s dose—a devastating scenario, particularly for patients with non-cancerous conditions like autoimmune diseases. However, complete removal of genetic material and other cellular debris remains challenging.
Given their direct contact with therapeutic NK cells, the EU imposes strict requirements on feeder cell use. Sandra Racordon-Pape, Director of Regulatory Science and Strategy at Cytiva, explained, “Under the ATMP GMP guideline (Part IV – Starting and Raw Materials), any material that comes into contact with cells must be of suitable quality, fully specified, traceable, and controlled in proportion to its potential impact on product quality and patient safety.”
These rules also apply to any NK product imported into the EU, effectively discouraging manufacturers that want access to the EU market from relying on feeder cell and driving united efforts toward feeder-free expansion strategies.
Animal-derived and human AB serums
Animal-derived growth factors like serum and supplements are another contributor to batch-to-batch variability and regulatory complexity (6). These serums support NK cell maintenance during expansion by providing hormones, vitamins, proteins, and growth factors (7). Fetal bovine serum (FBS) is widely used in clinical NK protocols as well as CAR T therapies (8).
Despite its common use, “FBS is treated as a high-risk critical raw material,” noted Sandra. “It can affect cell identity, potency, and safety, so regulators expect a risk-based control strategy with full origin traceability with a documented TSE/BSE assessment.” She emphasized that sterility, mycoplasma, and bovine virus testing must be predefined, and specifications for residual bovine proteins must be justified with clearance data and immunogenicity risk assessments.
“FBS is only acceptable when the submission demonstrates that product quality and patient safety are independent of the specific FBS lot,” she added. With FBS being prone to significant variation between lots (9), chemically defined media is becoming an increasingly attractive option across cell therapy modalities.
Scaling up NK cell therapy manufacturing
Addressing batch-to-batch variability is just one part of the equation. To realize NK cell therapy commercialization, large-scale manufacturing is essential. Current data suggests that high quantities of NK cells are needed for therapeutic efficacy, making scalable workflows a top priority across the industry.
Manual workflows and process inefficiencies
Despite the growing availability of automated cell therapy manufacturing platforms, recent reports highlight that current clinical NK workflows remain largely fragmented and depend on manual handling steps (10).
Manual intervention introduces risks familiar across cell therapy manufacturing: contamination, human error, and slow turnaround times. As processes scale, reliance on operators can create manpower bottlenecks and drive-up production costs.
For allogeneic therapies, automation will be key to scale up, reducing costs, and making treatments accessible and affordable for patients. Automated and closed manufacturing systems have helped CAR T manufacturers minimize the risk of batch failure and improve process efficiency. For NK cell therapies to reap the same benefits, cell processing systems must be able to support the large volumes required for a successful allogenic strategy.
Cryopreservation for allogenic therapies
Cryopreservation is also driving the need for large-scale NK cell expansion. While essential for delivering formulated doses to patients, only a fraction of cells retain cytotoxic function after thawing, with the quality of NK cells playing a factor in cell recovery (11).
Cryopreservation and thawing are also issues in the autologous world. However, the potency of CAR T means reduced cell numbers due to freezing and thawing can still induce a therapeutic effect on the patient. In contrast, traditional allogenic NK approaches require billions of cells in multiple doses for therapeutic impact.
Accounting for cell death from cryopreservation further increases the number of NK cells needed to provide a therapeutically effective dose to a patient, underscoring the critical need for robust expansion protocols that support allogenic-scale volumes.
Innovating for clinical and commercial success with NK cell therapy
CAR-NK: Enhancing tumor specificity
Chimeric antigen receptor NK cells (CAR-NK) are emerging as a next-generation cell therapy, combining NK cells’ innate cytotoxicity with the precision of CAR targeting. A more potent NK cell could lower the number of cells needed to see positive clinical outcomes, reducing the pressure on manufacturers to produce massive batches.
This exciting therapeutic approach has shown effective tumor targeting while maintaining the favorable safety profile of NK cells. Notably, clinical data so far has indicated low risk and severity of cytokine release syndrome, a life-threatening condition observed in some CAR T patients (14).
While autologous CAR T treatments have produced many patient success stories, donor variability of patient-derived material continues to be a challenge—one that CAR-NK cells may be positioned to overcome as an allogeneic therapy.
CAR-NK products can be produced from healthy donor NK cells or NK cell lines, including iSPC-derived cells. The potential to combine clonal iSPC cell lines (eliminating donor variability) with CAR engineering for high tumor specificity has some hoping for the next therapeutic breakthrough.
Dozens of CAR-NK therapies are under investigation worldwide, with encouraging early clinical results. For example, an off-the-shelf CAR-NK therapy targeting CD19 induced a one-year cumulative complete response rate of 83% in non-Hodgkin’s lymphoma patients, comparable to autologous CAR T outcomes, with no graft-versus-host disease and no high-grade cytokine release syndrome reported (4).
Automated, closed manufacturing systems
Closing and automating manufacturing workflows is essential to achieving batch-to-batch consistency required for efficient scale up. Planning for allogeneic commercial-scale production means transitioning to a scalable model as early as possible to avoid technology transfer complications later.
Difficulty expanding cells in a bioreactor after successful lab-scale trials is a familiar story across many cell therapy development projects, and it can be avoided by keeping the end goal of commercial scale-up in mind.
Fully integrated and automated platforms can streamline cell isolation, expansion, harvest, and cryopreservation by drastically reducing the number of handling steps and dependency on manual intervention. While dedicated platforms for NK cell processing have yet to reach the market, the following case study demonstrates how flexible and modular cell therapy manufacturing systems established for CAR T manufacturing have already been successfully adapted to help scale NK workflows.
Case study: Cell therapy manufacturing systems for NK production
Glycostem Therapeutics has made notable progress toward scalable, GMP-compliant NK cell manufacturing by adopting a semi-automated bioreactor process (15).
Monica Raimo, Senior R&D Manager for Glycostem Therapeutics, explained in a recent webinar that transitioning from a two-phase expansion, involving both static and bioreactor cultures, to using only the semi-automated Xuri™ bioreactor reduced the need for manual intervention and enabled perfusion-based feeding.
Monica continued, "Our combination of a semi-automated bioreactor-only process with improved, chemically defined medium led to higher yield, faster NK differentiation, and our process shortened to only 28 days to harvest larger quantities of highly functional NK cells."
These changes were also key to achieving the consistency required to optimize and standardize processes—critical steps for successfully navigating clinical trials. "Having systems like the Xuri™ bioreactor allows for standardization of your process parameters," added Jan Spanholtz, Chief Scientific Officer at Glycostem Therapeutics. Supporting both small and large batch sizes, the Xuri™ bioreactor enables continuity when scaling from early development to commercial-scale production.
With a leaner, semi-automated, and scalable process, Glycostem Therapeutics is advancing NK-based therapies through clinical trials. ONKord (Inaleucel) demonstrated an excellent safety profile and promising efficacy in Phase I trials for acute myeloid leukemia, with additional indications in pre-clinical development (16).
Watch the full webinar here.
Outlook and final thoughts
As NK cell therapy moves closer to clinical and commercial reality, manufacturing remains a critical hurdle. Addressing batch-to-batch variability, adopting closed and automated systems, and planning for scale early are essential to unlocking the full potential of allogeneic NK therapies.
The promise of allogeneic approaches lies not only in scientific innovation but in their ability to broaden access to advanced therapies, reaching patients beyond the limited number of centers equipped to deliver autologous CAR T therapies.
Emerging approaches such as feeder-free expansion, xeno-free chemically defined medium, CAR-NK engineering, and iPSC-cell lines are driving this innovation. When paired with robust bioprocessing strategies, off-the-shelf NK therapies have the potential to transform cancer treatment.
The question is no longer if NK therapies will reach patients, but how quickly we can overcome the manufacturing bottlenecks to make that future a reality.
FAQ
What makes allogeneic NK cell therapy different from autologous approaches?
Allogeneic NK therapies could be manufactured in advance and stored as off-the-shelf products, reducing logistical and timing constraints associated with patient-specific autologous cell collection and processing.
Why is batch‑to‑batch variability a major challenge in NK cell manufacturing?
Variability arises from donor‑derived starting material, open processing, and the use of animal and human-derived reagents, all of which introduce inconsistency in yield, purity, and regulatory risk.
Why do NK cell therapies require such large manufacturing volumes?
NK cells have shorter persistence and limited in vivo expansion compared to engineered T cells, meaning patients often require very high cell doses or multiple rounds of treatment.
What role does cryopreservation play in allogeneic NK therapy workflows?
Cryopreservation is essential for off‑the‑shelf distribution, but cell loss after thawing is significant, increasing the total number of NK cells required during production.
How close are NK therapies to commercial readiness?
Although no allogeneic NK therapy has yet received full FDA approval, several are in late‑stage clinical trials, with increasing emphasis on scalable, automated workflows to support commercialization.
References
- Colomar-Carando N, Gauthier L, Merli P, et al. Exploiting natural killer cell engagers to control pediatric B-cell precursor acute lymphoblastic leukemia. Cancer Immunol Res. 2022;10(3):291-302. doi:10.1158/2326-6066.cir-21-0843.
- Khani-Eshratabadi M, Motallebzadeh Khanmiri J, Dashti MR, et al. Potential therapeutic roles of natural killer cells in acute myeloid leukemia: a systematic review study. Clin Exp Med. 2025;25(1). doi:10.1007/s10238-025-01786-w.
- Cheng M, Chen Y, Xiao W, Sun R, Tian Z. NK cell-based immunotherapy for malignant diseases. Cell Mol Immunol. 2013;10(3):230–52. doi:10.1038/cmi.2013.10.
- Marin D, Li Y, Basar R, Rafei H, Daher M, Dou J, et al. Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: a phase 1/2 trial. Nat Med. 2024;30:772-784. doi:10.1038/s41591-023-02785-8.
- Motallebnejad P, Kantardjieff A, Cichocki F, Azarin SM, Hu WS. Process engineering of natural killer cell-based immunotherapy. Trends Biotechnol. 2023;41(10):1314-1326. doi:10.1016/j.tibtech.2023.03.018.
- Johnson CDL, Zale NE, Frary ED, Lomakin JA. Feeder-cell-free and serum-free expansion of natural killer cells using Cloudz microspheres, G-Rex6M, and human platelet lysate. Front Immunol. 2022;13:803380. doi:10.3389/fimmu.2022.803380.
- Lamers-Kok N, Panella D, Georgoudaki AM, Liu H, Özkazanc D, Kučerová L, et al. Natural killer cells in clinical development as non-engineered, engineered, and combination therapies. J Hematol Oncol. 2022;15:164. doi: 10.1186/s13045-022-01382-5.
- Eberhardt F, Hückelhoven‐Krauss A, Kunz A, et al. Impact of serum‑free media on the expansion and functionality of CD19.CAR T‑cells. Int J Mol Med. 2023;52(1). doi:10.3892/ijmm.2023.5261.
- Oikonomopoulos A, van D, Manansala AR, Lacey P, Tomakili TA, Ziman A, et al. Optimization of human mesenchymal stem cell manufacturing: the effects of animal/xeno-free media. Sci Rep. 2015;5:16570. doi: 10.1038/srep16570.
- Motallebnejad P, Kantardjieff A, Cichocki F, Azarin SM, Hu WS. Process engineering of natural killer cell-based immunotherapy. Trends Biotechnol. 2023;41(10):1314–26. doi:10.1016/j.tibtech.2023.03.018.
- Mark C, Czerwinski T, Roessner S, Mainka A, Hörsch F, Heublein L, et al. Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells. Nat Commun. 2020;11:5224. doi:10.1038/s41467-020-19094-0.
- Sun J, Elliott M, Souza‐Fonseca‐Guimaraes F. Engineered iPSC‐derived natural killer cells: recent innovations in translational innate anti‐cancer immunotherapy. Clin Transl Immunology. 2025;14(7). doi:10.1002/cti2.70045.
- Heipertz EL, Zynda ER, Stav-Noraas TE, Hungler AD, Boucher SE, Kaur N, et al. Current perspectives on “off-the-shelf” allogeneic NK and CAR-NK cell therapies. Front Immunol. 2021;12. doi:10.3389/fimmu.2021.732135.
- Huang, R, Wang, X, Yan, H et al. Safety and efficacy of CD33-targeted CAR-NK cell therapy for relapsed/refractory AML: preclinical evaluation and phase I trial. Exp Hematol Oncol. 2025;14(1). doi:10.1186/s40164-024-00592-6.
- Glycostem. Natural killer cells making better cancer immunotherapies. Biopharm Deal. 2020. www.nature.com/articles/d43747-020-00861-3. Accessed January 23, 2026.
- Dolstra H, Roeven MWH, Spanholtz J, et al. Successful transfer of umbilical cord blood CD34+ hematopoietic stem and progenitor-derived NK cells in older acute myeloid leukemia patients. Clin Cancer Res. 2017;23(15):4107–4118. doi:10.1158/1078-0432.CCR-16-2981.